Project Summary: The lack of proper biomarkers has greatly hampered investigations of the human health effects of the toxigenic fungus, Stachybotrys. Attempts to relate inhalation exposure to pulmonary, immune and neurological toxicities have had only circumstantial evidence supporting the apparent exposure. Epidemiological and pathophysiological studies would be greatly facilitated by the development of biomarkers that can be quantitatively related to the dose and timing of the exposure. We have recently found that purified satratoxin G, a macrocyclic trichothecene from Stachybotrys with 2 epoxides, forms stable adducts with human serum albumin. This offers the potential for quantitative biomarker 'dosimeters' in the form of protein adducts and related urinary metabolites in parallel to that used for the epoxide derivative of the mycotoxin, aflatoxin B1. Hypothesis: Inhalation exposure of humans to strains of Stachybotrys chartarum producing satratoxin G will result in the formation of covalent adducts of serum albumin and/or hemoglobin in sufficient quantities to be measured using mass spectrometry or radioimmune assay. This project proposes to: 1) Complete the identification of the amino acyl satratoxin adducts of recombinant human serum albumin, determine the chemical structure of the adducts, and ascertain the relative reactivity of the amino acyl residues being modified. 2) Confirm the presence of these adducts and/or metabolites in samples of biological materials including serum, red blood cells and urine from exposed rats and humans. 3) Develop practical quantitative biomarker assays for satratoxin-albumin and hemoglobin adducts using exhaustive proteolysis and immunoaffinity chromatography with analysis by mass spectrometry and by radioimmune assay. Similar mass spectrometry assays will be developed for related urinary metabolites. 4) Develop parallel assays for macrocyclic trichothecenes in environmental dust samples. Relevance: Over the past decade there has been increasing public concern about the health effects of "toxic mold" or "black mold" (i.e. Stachybotrys) in home and work environments. The lack of objective tools to relate the amount of exposure to the observed health problems has hampered attempts to clearly demonstrate such mold-related health effects. This project proposes to develop quantitative assays for such investigations. [unreadable] [unreadable] [unreadable]